| Project/Area Number |
18591180
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Pediatrics
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| Research Institution | University of Tsukuba |
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Principal Investigator |
ONODERA Masafumi University of Tsukuba, Graduate School of Comprehensive Human Sciences, Assistant Professor (10334062)
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| Co-Investigator(Kenkyū-buntansha) |
OKUYAMA Torayuki University of Tsukuba, Dept. of Clinical Laboratory Medicine/National Center for Child Health and Development, Director (40177192)
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| Project Period (FY) |
2006 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥3,190,000 (Direct Cost: ¥2,800,000、Indirect Cost: ¥390,000)
Fiscal Year 2007: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2006: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | gene / virus / immunology / translational research / regenrative medicine |
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| Research Abstract |
We have done experiments below to develop the new retroviral transduction system used in stem cell gene therapy clinical trials.
1) Cloning of the cytochrome b558 heavy chain (CYBB) gene: The target disease is X-linked chronic granulomatous disease (CGD) in which the CYBB gene is mutated and its coding protein gp9l^<phox> is non-functional, resulting in severe bacterial and fungal infections characterized by formation of granulomas. The open reading frame (ORF) of the gene was amplified by the PCR technique using cDNA reverse-transcribed from mRNA isolated from polynuclear cells of healthy control. The sequence of the ORF (1603bp) was certified.
2) Construction of the gene silencing resistant retroviral vector GCDNsap: GCDNsap was constructed by inserting mutations into the clinical retroviral vector Gcsap not to bind to the negative transcription factors such as YY1 and Trim28. The vector allows stable expression of the transduced genes in immature cells such as hematopoietic stem cells or embryonic stem cells.
3) Establishment of retroviral producer clones: The vector cloned with the CYBB cDNA was converted to the corresponding retrovirus by transduction into the 293gpg cell that expressed vesicular stomatitis virus derived G protein (VSV-G) under the control of tetracycline (tet off system). The virus titer was approximately 2.0x10^5 ml/I. U. assayed on Jurkat cell.
4) Expression of gp91^<phox> in transduced cells: Expression of the gene on the surface or cytoplasm of transduced cells was determined by FACS. Transduction experiments into human CD34^+ cells are under way
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